Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function

Abstract

The centrosome organizes microtubules by controlling nucleation and anchoring processes. In mammalian cells, subdistal appendages of the mother centriole are major microtubule-anchoring structures of the centrosome. It is not known how newly nucleated microtubules are anchored to these appendages. We show here that ninein, a component of subdistal appendages, localizes to the centriole via its C-terminus and interacts with gamma-tubulin-containing complexes via its N-terminus. Expression of a construct encoding the ninein C-terminus displaced endogenous ninein and the gamma-tubulin ring complex (gamma-TuRC) from the centrosome, leading to microtubule nucleation and anchoring defects. By contrast, expression of a fusion consisting of the N- and C-terminal domains (lacking the central coiled-coil region) displaced endogenous ninein without perturbing gamma-TuRC localization. Accordingly, only anchoring defects were observed in this case. Therefore, expression of this fusion appeared to uncouple microtubule nucleation and anchorage activities at the centrosome. Our results suggest that ninein has a role not only in microtubule anchoring but also in promoting microtubule nucleation by docking the gamma-TuRC at the centrosome. In addition, we show that the gamma-TuRC might not be sufficient to anchor microtubules at the centrosome in the absence of ninein. We therefore propose that ninein constitutes a molecular link between microtubule-nucleation and -anchoring activities at the centrosome.